Autoradiographs, or x-ray films, of nucleotide sequences are typically generated by a fundamental biochemical technique incorporating a process known as gel electrophoresis. Gel electrophoresis is a procedure that enables single-stranded DNA fragment molecules of a nucleic acid to be distinguished on the basis of size and/or charge. Electrophoresis is usually performed in a gelled (e.g., agarose) or polymerized (e.g., polyacrylamide) media (generically termed a "gel") that contains an electrically conducting buffer. Electrophoresis entails the application of a voltage via chemically inert metal electrodes across the cross-sectional area of the gel. The nucleic acid of interest is placed into pre-formed sample wells in the gel, usually at one end of the gel, and the polarity of the applied voltage is arranged so that the nucleic acid sample migrates through the gel towards one of the electrodes (usually positioned at the opposite end of the gel from the samples). Where appropriate, the inverse linear relationship between migration distance and molecular size is maintained by the addition of chemical denaturants (such as urea, formamide, or sodium dodecyl sulfate) to the gel and electrophoresis buffer.
Prior to conducting gel electrophoresis, a collection of single-stranded DNA fragments is generated either by chemical degradation of the nucleic acid (using the Gilbert method, see e.g., Maxam and Gilbert (1980), Methods Enzymeol., 65, 499-500) or most frequently by replacement DNA synthesis using a polymerase (using the Sanger method, see e.g., Sanger, F., Niklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467). This collection of single-stranded DNA fragments includes a fragment corresponding to each position in the sequence to be determined. Typically, this correspondence is directly related to the distance from a fixed site of initiation of polymerization at a primer that is annealed to the nucleic acid to be sequenced. Thus, determination of the desired sequence depends on the separation of each of the fragments, which differ in length by only a single nucleotide.
As further described in the '583 application, the identity of each of the four possible nucleotides at each position (adenine ("A"), guanine ("G"), cytosine ("C") or thymidine ("T")) is traditionally distinguished by performing a sequencing reaction specific for each ending nucleotide in a separate chemical reaction mixture. Typically, each of the four sequencing experiments is performed in a separate test tube. In each test tube, a collection of fragments is generated, each fragment ending at a position corresponding to the terminating nucleotide used in the given reaction. To determine the nucleotide sequence of the nucleic acid sample, gel electrophoresis is next performed on each of the four reaction mixtures, samples of the four reactions being electrophoresed individually in adjacent lanes of a single sequencing gel.
As is well known in the art, in order to perform electrophoresis, each sample to be electrophoresed is first loaded in a well at approximately one end of the sequencing gel. During electrophoresis, the gel is then in contact with electric current-carrying buffer solutions and is placed between two electrodes. Application of direct current voltage across the gel is achieved by placing the positive electrode at the end distal to the loading wells (also known as the "bottom end") and the negative end at the end proximal to the loading wells (also known as the "top end.") Electrophoresis is then achieved when the DNA molecules, or fragments, are separated in a direction going from the negative electrode to the positive electrode, wherein the smallest molecules travel the farthest toward the positive end. As a result, in theory, the presence of a band at a position in a nucleotide-specific lane of the gel indicates the identity of that nucleotide at that position in the sequence.
Conventionally, each of the fragments is radiolabeled, and, after electrophoresis, the four lanes and bands in the sequencing gel are visualized by autoradiography on x-ray films. As a result, the images on the resulting autoradiographs should theoretically be precise depictions of the band positions in the gel. Unfortunately, however, the ability to identify band positions on an autoradiograph of a DNA sequence has suffered from certain limitations.
One principle factor affecting the ability to determine the presence of a band at a given position in a nucleotide-specific lane (or, more specifically, in the combined nucleic acid sequence) is the band resolution. As indicated in the '583 application, band resolution depends on the thickness of each band as well as the relationship between the average thickness of each band and the width of the space separating each band. A sequencing ladder comprised of thick bands will contain fewer resolvable bands on average than a gel having thinner, more tightly-resolved bands, as a result of the finite length of the resolvable portion of the gel. Additionally, since each of the bands in a sequencing gel can in principle differ in size by as few as one nucleotide, stretches of DNA containing doublets, triplets or more multiple repeats of a particular nucleotide (e.g., 5'-TTTTTTTTT-3') have been difficult to resolve in a gel. In turn, therefore, it has been difficult to read such nucleotide sequences on the resulting autoradiograph x-ray film as well.
Another related factor affecting the ability to identify bases in a DNA sequence is the broadening of base bands. Higher up in the gel, where there are many close bands, the bands are sufficiently broad that they overlap each other. To the unaided human eye, these overlapping bands are seen as one broad band.
Yet another factor affecting the ability to read DNA sequences on autoradiographs is the nonlinearity of conventional sequencing gels and electrophoresis processes. In view of the above noted proportionality between DNA molecule size and distance traveled from the top end of the sequencing gel to the bottom end of the sequencing gel, theory would dictate that a linear distribution should occur along the entire length of the gel and, in turn, along the entire length of the autoradiograph. However, for a number of reasons, most electrophoresis gels and processes are structured such that the distribution of bases along any given nucleotide-specific lane in the gel and on the resulting autoradiograph is nonlinear over a portion of the gel, such nonlinearity typically being most pronounced in the region of the gel most proximal to the loading well. As is known in the art, this most nonlinear region of the gel proximal to the loading well is sometimes referred to as the "reptation regime." In contrast, the more linear region approaching the end most distal to the loading wells, is sometimes referred to as the "Ogston regime" or "sieving regime."
The substantial nonlinearity in the reptation regime, and, to a lesser extent, in the Ogston regime, results at least in part from the loading process, from thermal diffusion and from gel nonuniformity. More particularly, as a result of these factors, autoradiographs of single-stranded DNA sequences have been observed to have relatively tightly spaced bands at the top end and rather widespread bands at the bottom end. Thus, while it has been relatively easy to read and identify the bases at the extreme bottom end of an autoradiograph, it has been relatively difficult if not impossible to identify the bases approaching the top end of the autoradiograph. This difficulty in identifying bases at the top of the autoradiograph is exacerbated at occurrences of doublets, triplets or other multiple repeated nucleotides as described above.